The processes to obtain economically relevant compounds have been changing significantly in recent years in the area of biotechnology. Until recently, the traditional industrial fermentations, for instance, were the main technological option for the production of polypeptides, enzymes, antibiotics and other substances of economic interest. Meanwhile, as the knowledge about microbiology, biochemistry and genetics of the organisms involved in the fermentative processes increased, the production technologies have also been adapted and modified. The development of molecular biology techniques was remarkable in this context: besides offering tools for the understanding of the biochemical, genetic and evolutionary mechanisms of the many species studied, molecular biology also furthered the development of countless alternatives for the industrial production of substances, as a result of the combination possibilities of the different organism's characteristics.
The present-day market shows a great demand of products and processes in the Human and/or Animal Health segment and the interest in the development of technologies in this area continues. From the industrial technology standpoint, many factors can be considered as limiting or technological “bottlenecks”. Especially relevant in the production of economically important substances are the costs involved in the purification, which frequently demands a series of complex and costly steps. The determination of the cost of new therapeutic products is a very complex process, if all phases involved are to be considered—from the product conception until its placement on the market. One major factor of impact on the costs is the adopted technology, especially in the case of biological products whose chemical synthesis has not yet been developed or which presents technical or economical disadvantages. The advance of the recombinant DNA technology allowed the production of proteins and other substances in virtually unlimited quantities. On the other hand, the expression system employed has a remarkable influence on the product's nature and on the production process. A good illustrative example is the production of the Tissue Plasminogen Activator (tPA), an anti-thrombotic agent used in the treatment of myocardium infarct, thrombosis and pulmonary embolism. This substance, when produced by Escherichia coli, or through the cultivation of genetically modified ovary cells from hamsters (CHO), presents significant differences regarding its structure (glycosilation, need of renaturing etc.) and production processes, which reflects on its price. In E. coli, 12% of the tPA's production costs correspond to the fermentative phase of the process and 88% are due to purification. On the other hand, in the production through CHO, 75% of the costs derive from the cellular cultivation and 25% from purification (Datar et al, “Process Economics of Animal Cells and Bacterial Fermentations: A Case Study Analysis of Tissue Plasminogen Activator” Bio/Technology 11:349-357, 1993). These differences are significant and can be decisively influent in the choice of the process to be used. Still other factors have a special importance in the choice of the production system, such as funds (capital) for the factory's installation, costs of raw materials, the need of qualified personnel (and its cost) as well as environmental and safety aspects.
The Vegetal Model
A safer and cheaper system to produce biologically originated substances may be idealized in transgenic plants cultivated in contained areas (greenhouses) or in agricultural areas. Furthermore, the vegetal system to produce substances of pharmaceutical interest offers various advantages, among which one might mention the absence of animal viruses and other animal cell products, as well as the absence of the typical contamination of bacterial fermentative processes, from yeasts or animal cells. In this context, the knowledge of the genetic and biochemical mechanisms of some vegetal models is reasonably wide, standing out, among other plants, Arabidopsis thaliana, Nicotiana tabacum and Oryza sativa. Among the main advantages of the use of the vegetal system for the production of substances with economical interest, one should emphasize the easier scale up, which is fundamental in the case of polypeptides and/or proteins of industrial interest. In the case of using microorganisms (recombinant or not), the phases involved in the scale up are normally the limiting factors of the process economy. This occurs for various reasons, among which the non-linearity of the determining relations of the process' efficiency (oxygen transfer, rheological factors, energy demand of the process etc.), the cost of the necessary production equipment and the need of adequately qualified personnel. In the case of the vegetal system, the mentioned cost components are much lower, since the scale-up tends to be simpler and linear. Besides that, in the evaluation of an industrial process' economy, the strategies seeking to eliminate the largest possible number of purification phases should be considered. Furthermore, plants are metabolically able to perform complex post-translational modifications, such as glycosylations, which widens the scope of possible peptides, antigens or vaccine candidates to be produced by plants. In this sense, the strategy of the present invention comprises the development of expression systems which take into account the most appropriate cellular and sub-cellular localizations for the desired products and, foremost, the elimination of purification steps.
The reproductive systems of A. thaliana and N. tabacum have been intensely studied in the recent years and are suitable to the logic of eliminating purification steps of the present invention. The starting point for the idealization of the present invention was the existing knowledge about the genes involved in the vegetal reproductive development and, more precisely, those related to the development of the tapetum which, for some years now, is the study object of the Laboratory for Plant Molecular Genetics of the Federal University of Rio de Janeiro, Brazil. Former studies in that laboratory in this area comprise the state of the art of the present invention. On one hand, the knowledge about the expression of the genes involved in the formation of A. thaliana's inflorescence (Franco, MSc. Dissertation, UFRJ, 1992) was helpful to understand the function of the oleosin-type proteins, more recently studied with molecular biology techniques associated to microscopy (Ferreira, PhD Thesis, UFRJ, 1997). On the other hand, there also exists knowledge about the regulation mechanisms of the codifying genes of said oleosin-type proteins, studied with the help of the .beta.-glucuronidase (GUS) gene marker (Scholte, PhD Thesis, UFRJ, 1998). Even more recently, a strategy of modifying the protein composition of the external surface of pollen grains was described by Foster et al “Modifying the pollen coat protein composition in Brassica”, Plant Journal 31(4): 477-486, 2002, also described in the document WO 99/49063, by the same authors. However, neither these, nor any other reference known by the author make any allusion or suggestion regarding the use of genetically modified plants for the production of pharmaceutical products in tissues and/or cells of the male vegetal reproductive system, neither do they mention the use of whole, intact, pollen grains derived from genetically modified plants as pharmaceutical products to be used in immunoreactions, as vaccines or as reactive agents for diagnostics, which collectively comprise the objects of the present invention.
For the purposes of the present invention, one should understand as “tissues and cells of the male vegetal reproductive system” the tissues or cells of the male vegetal reproductive system, including the anthers, tapetum, pollen—grains, parts and combinations thereof. For the purposes of the present invention, one should also understand as “immunoreactions” all reactions that involve cells and/or molecules of the immune system of eukaryotes, including vertebrates, invertebrates, mammals and the like, including mononuclear cells such as macrophages and lymphocytes B and T, neutrophils, eosinophils, besides antigens, antibodies, cytokines and other chemical mediators of the immune system, including parts of the same and its combinations. Furthermore, for the purposes of the present invention, one should understand as “heterologous polypeptide” any amino acid sequence which is not naturally produced by the plant, but whose synthesis in it derived from the genetic modification undertaken in the plant through the present invention. Without limiting the scope of the present invention, one should emphasize as being of special importance the use of pollen grains containing at least one heterologous polypeptide.
The present invention offers means to avoid some difficulties in both the production and use of polypeptides with therapeutic and/or diagnostics interest. On one hand, pollen grains are structurally stable, probably as a consequence of the need of reproductive success. Therefore, their “evolution” in order to resist the most diverse environmental stresses is useful to the logic of using them as a product, since their high stability is favorable and desirable. In an additional aspect, whole pollen grains of the present invention may be used in certain applications, bypassing the need of purification. As examples, herein used to illustrate the present invention but not to limit its scope, whole pollen grains containing heterologous polypeptides would permit: the direct use as antigens in the production of diagnostic kits, especially for screening, and its use as a vaccine preferably delivered onto mucous membranes or injected subcutaneously.
Pollen is often associated to allergy because some people develop symptoms such as sneezing, itching, cough, nasal irritation, eye watering and asthma when exposed to the pollen of certain plants. These particles are carried in large amounts by the air, normally during springtime. When they get in contact with the nasal mucous membranes or the throat, they may trigger allergic reactions known as polinosis or seasonal allergic rhinitis (for more details, consult Balda et al, “Tree-pollen Allergy is Efficiently Treated by Short-term Immunotherapy (STI) with Seven Preseasonal Injections of Molecular Standardized Allergens”. Allergy 53, 740-748, 1998). As in any allergic process, the polinosis is a high sensibility to certain substances present in the pollen, and considerably varies from person to person, even though there seems to be a familiar correlation. In the majority of allergic reactions, the immune system responds to a “false alarm”, mobilizing the attack against the allergen. The organism produces large quantities of specific IgEs, which bind themselves to the mastocytes in the tissues and to the basophils in the blood. When the allergen meets the IgE the liberation of histamine, prostaglandin, leucothriens and other substances occurs, thus causing the allergy symptoms (for more details, see Batanero et al, “IgE-binding and Histamine-release Capabilities of the Main Carbohydrate Component Isolated from the Major Allergen of Olive Tree Pollen, Ole e 1”. J Allergy Clin Immunol 103, 147-153, 1999). Some strategies have been developed in order to obtain vaccines against autoimmune diseases and allergies, being generally based on the induction of tolerance. For example, known pollen grain allergens, when prepared in an encapsulated form, have shown to be efficient in the induction of tolerance by means of nasal administration. Nevertheless, the available encapsulating methods of allergens/antigens are laborious and costly, besides generally bringing about the denaturation of said allergens/antigens. The present invention's approach makes it possible to overcome these problems through the production of heterologous allergens/antigens in pollen grains.
Due to its natural ability to stimulate the production of specific IgEs and IgGs, we started from the hypothesis that the pollen could become a good candidate as a carrier and deliverer of vaccine antigens in mucous membranes, such as its direct use via nasal immunizations. Besides the already mentioned elevated structural stability of the pollen, an additional characteristic of the present invention under consideration is to enable the use of whole pollen grains as vaccines. Interestingly, one of the goals clearly expressed by the World Health Organization (WHO) is the development of new systems for the delivery of antigen vaccines to the respiratory tract. Furthermore, recent studies have demonstrated that vaccines derived from transgenic plants, when applied in the form of dry powder, seem to be the preferred solution for the lack of homogeneity in the concentrations of antigens produced in plants (for further references, see Sala et al., “Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives”. Vaccine 21:803-808, 2003; Mielcarek et al. “Nasal vaccination using live bacterial vectors”. Advanced Drug Delivery Reviews 51:55-69, 2001). On the other hand, the WHO's recommendation for the vaccine against tetanus (or lockjaw), for instance, is the administration of three consecutive doses of the respective antigen. This repeated administration has financial and logistical disadvantages, since some patients do not return for the second dose and because the vaccination campaigns have as a limiting factor: the need of a cold chain, considering that practically all vaccines are thermo labile. The availability of vaccines in pollen grains presents itself as capable of solving these mentioned problems, besides being easily delivered (without injections), easily standardized and endowed with an elevated thermal stability, being able to help avoid the logistics problems which are so far inherent to vaccination campaigns. These advantages open up good perspectives for the study and development of this way of immunization in combination with the presentation form of the present invention.
Several efforts have been made in the last two decades in the sense of trying to develop subunit vaccines for human and veterinary use. The subunit vaccines are based on individual components derived from the infectious agent and, normally, have a low immunogenicity due to the absence of other cellular constituents from which they are often purified. Therefore, when developing vaccines it is desirable to plan the utilization of other substances which have the potential to increase the immune response to the antigens in question, what is normally done with the use of adjuvants. Entire cells or parts thereof can work as self-adjuvants, which is favorable for the present invention's approach. The identification of an appropriate antigen is only the first step in the development process to obtain a subunit vaccine, since adequate adjuvant systems and delivery systems of the respective antigen are also necessary. An adjuvant can be any material which increases the immune-humoral and/or cellular response to the antigen(s); it is generally accepted in literature that certain adjuvants act through the gradual liberation of the antigens to the cells of the immune system. Recent studies (Wiedermann, et al. “Modulation of an allergic immune response via the mucosal route in a murine model of inhalative type-I allergy” Intl. Arch. Allergy Immunol. 118:129-132, 1999) have shown that antigenic preparations in powdered form can also increase the incorporation of antigens by the antigen processing cells of the immune system. In this sense, the use of whole pollen grains, due to its powdered nature, offers this additional advantage in the case of application in vaccines. Independently of exactly knowing the specific mechanism involved, it is known that not only the cellular, but also the humoral immunity might be stimulated in various degrees, depending on the antigen, the adjuvant, the administration protocol and the species involved.
In order to develop an effective and commercially feasible vaccine, the relation between the production cost and the large-scale production capacity of the antigenic preparation and the adjuvant system should be taken into account. On the other hand, the growing number of vaccines under developed and the number of required injections for a wide-ranging immunization program for children, for instance, generates elevated costs and the preoccupation about the discontinuity potential of current vaccination programs. This makes highly desirable the availability of alternative immunization means, especially those allowing the so-called multivalent vaccination, that is, the one that permits to present multiple antigens or epitopes simultaneously. In this context, transgenic plants have been the object of several attempts to obtain “edible” vaccines, due to their notorious advantages in terms of production costs and administration. However, vaccines produced in transgenic plants destined for oral consumption have the disadvantage of passing by the gastrointestinal barrier, which destroys a significant portion of the vaccine antigens. For that reason, the state of the art shows that the amount of antigens required for an effective immune response derived from an edible transgenic plant vaccine is from 100 to 1,000 times higher than the amount of antigens necessary for an effective parenteral immunization (for further reference, see Carter III, J. E., and Langridge, W. H. R. “Plant-based vaccines for protection against infectious and autoimmune diseases”. Critical Reviews in Plant Sciences 21(2), 93-109, 2002; Streatfield and Howard “Plant-based vaccines”. International Journal for Parasitology 33, 479-493, 2003). Therefore, the need to develop new alternatives to overcome these difficulties persists.
The entry route of most pathogenic agents is the mucous surfaces and a large part of the infections is located in the mucous and sub mucous tissues. However, conventional injectable vaccines are poorly efficient for the induction of an immune response in mucous membranes. As a consequence, several experiments are being carried out in order to offer alternatives for the immunization in mucous membranes, which includes, for instance, the incorporation of antigens in larger particles (such as liposomes, immune stimulatory complexes, microspheres) for increasing the efficiency of the immune response in mucous tissues. On the other hand, the use of adequate adjuvants is normally very important for the stimulation of the desired type of immune response. Among the known adjuvants, and in the scope of the present invention, one might point out the vegetable oils. A primary advantage of the use of vegetable oil adjuvants over the use of mineral oils is that vegetable oils can be easier metabolized and are, therefore, more tolerable. Patent literature is rich in examples of the use of vegetable oils as adjuvants in vaccines, including, for instance, documents WO 01/95934 “The use of plant oil-bodies in vaccine delivery systems” and WO 02/00169 “Production of vaccines using transgenic plants or modified plant viruses as expression vectors and transencapsidated viral coat proteins as epitope presentation systems”. Most of these patents disclose formulations and the use of water-in-oil or oil-in-water emulsions which are prepared through the mixture of pure chemical compounds. However, none of the mentioned documents reveals or suggests the use of tissues or cells from the male vegetal reproductive system, such as whole pollen grains, or even parts thereof, as an useful pharmaceutical product in immune reactions or as an antigen-adjuvant combination for immunization, preferably applicable in mucous membranes. In this context, the present invention offers a technical and commercially feasible production alternative of a pharmaceutical product which is potentially useful in the treatment of dysfunctions of the immune system of eukaryotes and which allows, among other characteristics, the production of multiple substances in one single system.